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Real-time visualization of complexin during single exocytic events

Nature Neuroscience volume 13, pages 577–583 (2010)Cite this article

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Abstract

Understanding the fundamental role of soluble NSF attachment protein receptor (SNARE) complexes in membrane fusion requires knowledge of the spatiotemporal dynamics of their assembly. We visualized complexin (cplx), a cytosolic protein that binds assembled SNARE complexes, during single exocytic events in live cells. We found that cplx appeared briefly during full fusion. However, a truncated version of cplx containing only the SNARE-complex binding region persisted at fusion sites for seconds and caused fusion to be transient. Resealing pores with the mutant cplx only partially released transmitter and lipid probes, indicating that the pores are narrow and not purely lipidic in structure. Depletion of cplx similarly caused secretory cargo to be retained. These data suggest that cplx is recruited at a late step in exocytosis and modulates fusion pores composed of SNARE complexes.

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Figure 1: Imaging secretory granules labeled with NPY-mRFP undergoing exocytosis in PC12 cells coexpressing different versions of cplx-GFP.
Figure 2: Effect of cplx overexpression on NPY-mRFP exocytosis.
Figure 3: Slow and incomplete release of NPY-mRFP when cplx persists during fusion.
Figure 4: Rapid resealing of granules with persisting cplx.
Figure 5: Restricted lateral diffusion of FM dyes with persisting cplx.
Figure 6: NPY-mRFP exocytosis in cells depleted of cplx.
Figure 7: Reduced transmitter release with persisting cplx.

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Acknowledgements

We thank F. Rivera-Molina and S. Viviano for technical assistance and P. De Camilli and D. Toomre for comments on the manuscript. This work was supported by McKnight and Kinship Foundations and US National Institutes of Health grants EY000785 and EY018111.

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  1. Chad P Grabner

    Present address: Present address: University of Saarland, Institute for Physiology, Homburg/Saar, Germany.,

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  1. Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA

    Seong J An, Chad P Grabner & David Zenisek

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S.J.A. designed, conducted and analyzed the research and wrote the manuscript. C.P.G. performed and analyzed the amperometry experiments. D.Z. supervised the project.

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Correspondence to Seong J An.

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An, S., Grabner, C. & Zenisek, D. Real-time visualization of complexin during single exocytic events. Nat Neurosci 13, 577–583 (2010). https://doi.org/10.1038/nn.2532

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